Optimized antigen-binding constructs of A33, an antibody that binds to colon cancer, have been engineered to be non-immunogenic;retain antigen-binding ability, and clear faster from non-tumor sites. Current immunoglobulin-based therapy of colon cancer as well as other solid tumors is limited by low tumornontumor ratios. We have undertaken an effort to increase this ratio by a) increasing serum clearance;b) increasing tumor penetrance of antigen-binding construct, and c) developing bispecific antigen-binding constructs that recognize tumor antigen and chelates that can carry radionuclides. This grant aims to study two antigen-binding constructs: a single chain Fv (sFv) fragment, and a fusion protein consisting of sFv A33 and a sFv against a chelate (DOTA). The clinical trials will be carried out in patients with measurable disease: the initial trials in patients scheduled to undergo biopsy of their disease, followed by trials using positron-emitting radiometals and serial PET imaging. These studies will help determine the optimum antigen-binding construct for subsequent radioimmunotherapy. Specific Aim 1 will determine the tumor targeting abilities and optimum route of administration of sFv A33 in patients with measurable metastatic colon cancer. The first study will be with IV 111ln-DOTA-sFv A33 in presurgical patients with metastatic colon cancer;if this study demonstrates targeting to normal colon and/or tumor, we will carry out a comparable study with intra-arterial 111ln-DOTA-sFv A33, to determine whether route of administration influences targeting. These will be followed by a PET study with 86Y-DOTA-sFv A33. We are in the process of producing a bispecific antigen-binding construct consisting of 2 sFv molecules: one sFv targets the A33 antigen while the other sFv reacts with metal-conjugated DOTA chelate. Specific Aim 2 will determine the optimal route of administration and targeting of this construct, with clinical trial design being similar to that in Specific Aim 1. Specific Aim 3 will evaluate tumor targeting using PET, and immunogenicity of a novel humanized A33 IgG. These studies will form the template for studies to determine the utility of sFv A33 in the detection and treatment of colon cancer;the utility of bispecific constructs in the detection and treatment of colon cancer; and the ability of a novel non-immunogenic A33 IgG to specifically target colon cancer.